The long term goal of this research project is to understand how neuronal growth cones reach their target sites in the vertebrate central nervous system (CNS). This is a fundamental problem in neuroscience. The delineation of how this is done should be important for understanding what goes wrong during developmental diseases of the nervous system and why regeneration following injury or disease to the CNS is normally so poor. Growth cones reach their targets by selecting appropriate pathways and by extending in the correct direction on these pathways. This proposal seeks to analyze how growth cones know what direction they should extend in the vertebrate brain. Although many experiments have demonstrated that growth cones can distinguish appropriate pathways from inappropriate ones relatively little is known about the nature of directionality cues. Past experiments suggest that directional information is provided by extrinsic polarity cues that are likely encoded as a gradient of molecules distributed along an axis. However, it is not known whether a system of widely distributed and orthogonally arrayed (dorsal/ventral and anterior/posterior) polarity cues specify direction for all growth cones in the vertebrate CNS. The zebrafish embryo may be especially useful for analysis of these issues since it is a vertebrate embryo that can be analyzed with a combination of cellular, molecular, and genetic techniques. This proposal seeks to systematically examine how growth cones know direction by transplanting neurons to ectopic sites and by time lapse analysis of their growth cones. The proposed experiments take advantage of the ability to precisely manipulate even single cells with the ability to observe living growth cones in zebrafish embryos. These experiments should determine whether polarity cues guide growth cones, how the cues are distributed, how many cues exist, how growth cones behave under the action of polarity cues, and possibly identify sources of polarity cues. In the future molecular analysis and the emerging genetics of zebrafish may identify candidate polarity genes for growth cones in the zebrafish. A detailed understanding of polarity cues at the cellular level should then be invaluable for the functional analysis of potential growth cone polarity genes in the zebrafish.